Project # 3: Molecular and Cellular Mechanisms of Presynaptic activity Long-lasting neural circuit modifications are thought to underlie all forms of experience-dependent plasticity, including learning and memory. Thus, there has been great interest in elucidating the mechanisms and functions of various forms of synaptic plasticity. While excellent progress has been made in elucidating the mechanisms of postsynaptic plasticity little is known about how presynaptic forms of plasticity are encoded. Recent genetic and physiological studies on the presynaptic active zone protein RIM have demonstrate that this molecule plays essential roles in the regulated release of neurotransmitter. Furthermore, RIM has been implicated in regulating a special type of cAMP dependent long-lasting increase in neurotransmitter release. However, mechanistically it is unclear how RIM accomplishes these diverse functions. Structural studies support a model wherein RIM promotes presynaptic plasticity through its ability to interact with a number of key active zone proteins including Munc13, a critical SV priming factor. In this project, we propose to explore whether RIM isoforms regulate neurotransmitter release probability by dynamically controlling the levels of Mund3 tethered to the active zone cytoskeletal matrix. This will be accomplished by addressing the following issues. In aim #1, we will define the cellular and molecular correlates of presynaptic plasticity in dissociated neuronal cultures. In aim # 2, we will dissect how RIM1a and its specific domains contribute to setting SV release probability. Finally in aim #3, we will assess the functional significance of RIM2 isoforms to presynaptic function? Furthermore, close collaborations with project #1 and #2 will explore the structural and physiological importance of discrete domains in RIMs and their interacting proteins for synaptic plasticity. Similar collaborations with project #4 will investigate whether RIM regulates the dynamic states of synaptic proteins at subclasses of synapses in the cerebellar system. These studies will advance our understanding of the mechanisms encoding synaptic plasticity.